The goal is to identify genetic events which enable diploid human fibroblasts to acquire neoplasia-associated phenotypes, principally anchorage-independent growth. We propose to do experiments involving the transfer of activated oncogenes into human fibroblasts as well as experiments involving the molecular characterization of BP anti diol-epoxide-induced, anchorage-independent human fibroblasts. These experiments will be done in order to identify relevant human transforming genes. Complementation of these discrete genetic events will be studied in an attempt to understand the genetic basis of the tumorigenic transformation of human fibroblasts. Several technical approaches will be used in these experiments, these include: transfection or electroporation of cloned oncogenes into human fibroblasts; mutagen induction of anchorage-independent growth in human fibroblasts and gene-specific DNA and RNA hybridization to identify gene amplification/over-expression events; screening DNA from anchorage-independent human fibroblasts for its ability to tumorigenically transform NIH/3T3 cells and subsequent cloning of the transforming gene by a cosmid rescue technique; screening DNA from anchorage-independent human fibroblasts for its ability to induce anchorage-independent growth in recipient human fibroblasts and cloning of this gene within a selectable cosmid which replicates extrachromosomally in human fibroblasts. The cloned NIH/3T3 and human transforming genes, as well as their wild-type homologues from a human DNA library, will be subjected to DNA sequence analysis. Our goal is to relate mutagen-induced acquisition of cancer-related phenotypes, such as anchorage-independence in human cells, to molecular changes at specific gene loci in order to understand transformation of human fibroblasts.